Method of treating viral hemorrhagic fever with activated protein C

ABSTRACT

The present invention provides a method of treatment of viral hemorrhagic fever with activated protein C. The claimed invention provides a needed therapy for a serious and debilitating disorder while avoiding complications such as bleeding tendency, toxicity and general side effects of currently available anti-coagulant agents.

This application claims priority of Provisional Application Ser. No.60/109,153 filed Nov. 20, 1998.

FIELD OF THE INVENTION

This invention relates to medical science particularly the treatment ofviral hemorrhagic fever with protein C.

BACKGROUND OF THE INVENTION

Protein C is a vitamin K dependent serine protease and naturallyoccurring anticoagulant that plays a role in the regulation ofhemostasis by inactivating Factors Va and VIIIa in the coagulationcascade. Human protein C circulates as a 2-chain zymogen, but functionsat the endothelial and platelet surface following conversion toactivated protein C (aPC) by limited proteolysis with thrombin incomplex with the cell surface membrane protein, thrombomodulin.

In conjunction with other proteins, aPC functions as perhaps the mostimportant down-regulator of blood coagulation resulting in protectionagainst thrombosis. In addition to its anti-coagulation functions, aPChas anti-inflammatory effects through its inhibition of cytokinegeneration (e.g. TNF and IL-1) and also exerts profibrinolyticproperties that facilitate clot lysis. Thus, the protein C enzyme systemrepresents a major physiological mechanism of anti-coagulation,anti-inflammation, and fibrinolysis.

Viral hemorrhagic fever is a clinical syndrome associated withsignificant mortality. Without exception, hemorrhagic fever viruses areenveloped RNA viruses that belong to four viral families: Arenaviridae[Junin, Machupo, Lassa fever], Bunyaviridae [Crimean-Congo hemorrhagicfever, Rift Valley fever, Hantaan and related viruses], Filoviridae[Ebola, Marburg] and Flaviviridae [Dengue, Yellow fever, Omskhemorrhagic fever, Kyasanur Forest disease], [Cosgriff, T. M., Reviewsof Infectious Diseases 11(4): S672-S688, 1989]. These agents produce awide spectrum of disease severity, but the most extreme manifestationsinclude circulatory instability, increased vascular permeability, anddiffuse hemorrhage [Lacy, et al. Advances in Pediatric InfectiousDiseases, 12:21-53, 1997].

The underlying mechanism of the bleeding in the hemorrhagic fevers iscomplex. Possible factors include thrombocytopenia alone, orthrombocytopenia associated with disseminated intravascular coagulation(DIC). Central to the mechanism may well be endothelial celldysfunction, which has profound implications for both platelets andcoagulation. Another possible factor is a decrease in levels ofcoagulation factors in plasma as the result of either increasedconsumption or impaired synthesis. Increased consumption occurs in DIC,while impaired synthesis is the likely consequence of liver injury.Liver involvement is a universal occurrence in viral hemorrhagic fever.For example, in Yellow fever, Rift Valley fever and Crimean-Congohemorrhagic fever, the temporal association of hemorrhage with severhepatic dysfunction is evident.

Viruses alter hemostasis in two general ways. The first is throughdirect effect on cellular functions, and the second is throughactivation of immune and inflammatory pathways. Both mechanisms may leadto variable degrees of cellular injury, including cell death. Activationof coagulation pathways is an important part of immune and inflammatoryreactions and accounts for the fibrin deposition that sometimes isobserved in these reactions.

Thrombocytopenia is a universal occurrence in viral hemorrhagic fevers.For example, in dengue hemorrhagic fever both changes suggestive ofdecreased thrombopoiesis and of increased platelet consumption has beendetermined. This is also the case in hemorrhagic fever with renalsyndrome (HFRS) caused by Hantaan and related viruses. Other mechanismsfor increased platelet destruction in viral infections include directinteraction of platelets with viruses, DIC, and endothelial injury.

In Ebola and Marburg hemorrhagic fevers, generalized hemorrhages arefound in most organs. Focal necrosis without significant inflammation isalso widely seen, especially in the lungs, liver, kidneys, and lymphoidorgans. DIC is common.

Currently, there is no effective therapy to treat viral hemorrhagicfever. In the absence of viral-specific chemotherapy, management isprimarily supportive. Therefore, a need exists for a safe, effectivetherapy of patients with viral hemorrhagic fever.

The present invention is the first to describe the treatment of viralhemorrhagic fever with protein C. Protein C, with its anticoagulant,anti-inflammatory, and profibrinolytic activities, is useful for thetreatment of the hypercoagulable state or protein C deficiency thatoccurs in viral hemorrhagic fever patients.

SUMMARY OF THE INVENTION

The present invention provides a method of treating a patient sufferingfrom viral hemorrhagic fever which comprises, administering to saidpatient a pharmaceutically effective amount of protein C.

The present invention further provides a method of treating viralhemorrhagic fever in a patient in need thereof, which comprisesadministering to said patient a pharmaceutically effective amount ofactivated protein C such that an activated protein C plasma level ofabout 2 ng/ml to about 300 ng/ml is achieved.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of the present invention, as disclosed and claimed herein,the following terms are as defined below.

Protein C refers to a vitamin K dependent serine protease withanticoagulant, anti-inflammatory, and profibrinolytic properties whichincludes, but is not limited to, plasma derived and recombinant producedprotein C. Protein C includes and is preferably human protein C althoughprotein C may also include other species or derivatives having protein Cproteolytic, amidolytic, esterolytic, and biological (anticoagulant,pro-fibrinolytic, and anti-inflammatory) activities. Examples of proteinC derivatives are described by Gerlitz, et al., U.S. Pat. No. 5,453,373,and Foster, et al., U.S. Pat. No. 5,516,650, the entire teachings ofwhich are hereby included by reference.

Zymogen—an enzymatically inactive precursor of a proteolytic enzyme.Protein C zymogen, as used herein, refers to secreted, inactive forms,whether one chain or two chains, of protein C.

Activated protein C or aPC refers to protein C zymogen which has beenconverted by limited proteolysis to its activated form. aPC includes andis preferably human protein C although aPC may also include otherspecies or derivatives having protein C proteolytic, amidolytic,esterolytic, and biological (anticoagulant or pro-fibrinolytic)activities. Examples of protein C derivatives are noted above in thedescription of protein C.

HPC—human protein C zymogen.

r-hPC—recombinant human protein C zymogen.

r-aPC—recombinant human activated protein C produced by activating r-hPCin vitro or by direct secretion of the activated form of protein C fromprocaryotic cells, eukaryotic cells, and transgenic animals or plants,including, for example, secretion from human kidney 293 cells as azymogen then purified and activated by techniques well known to theskilled artisan and demonstrated in Yan, U.S. Pat. No. 4,981,952, andCottingham, WO97/20043, the entire teachings of which are hereinincorporated by reference.

Plasma derived activated protein C—activated protein C produced byactivating plasma HPC as described in Eibl, U.S. Pat. No. 5,478,558, theentire teaching of which is herein incorporated by reference.

Continuous infusion—continuing substantially uninterrupted theintroduction of a solution into a vein for a specified period of time.

Bolus injection—the injection of a drug in a defined quantity (called abolus) over a period of time up to about 120 minutes.

Suitable for administration—a lyophilized formulation or solution thatis appropriate to be given as a therapeutic agent.

Unit dosage form—refers to physically discrete units suitable as unitarydosages for human subjects, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

Pharmaceutically effective amount—represents an amount of a compound ofthe invention that is capable of inhibiting sepsis in humans. Theparticular dose of the compound administered according to this inventionwill, of course, be determined by the attending physician evaluating theparticular circumstances surrounding the case.

Viral hemorrhagic fever—refers to hemorrhagic fever caused by envelopedRNA viruses that belong to four viral families: Arenaviridae [Junin,Machupo, Lassa fever], Bunyaviridae [Crimean-Congo hemorrhagic fever,Rift Valley fever, Hantaan and related viruses], Filoviridae [Ebola,Marburg] and Flaviviridae [Dengue, Yellow fever, Omsk hemorrhagic fever,Kyasanur Forest disease]. These agents produce a wide spectrum ofdisease severity, the most extreme manifestations includes circulatoryinstability, increased vascular permeability, and diffuse hemorrhage.

The present invention provides for the treatment of viral hemorrhagicfever with protein C. Protein C, with its anticoagulant,anti-inflammatory, and profibrinolytic activities, is useful for thetreatment of the hypercoagulable state and/or protein C deficiency thatoccurs in viral hemorrhagic fever patients.

The protein C administered according to this invention may be generatedand/or isolated by any means known in the art or as described in U.S.Pat. No. 4,981,952, and U.S. Pat. No. 5,550,036, herein incorporated byreference. For example, protein C can be produced by secretingfull-length, soluble protein C, or biologically active polypeptidevariants of protein C from a cell which comprises (a) constructing avector comprising DNA encoding protein C; (b) transfecting the cell withthe vector; and (c) culturing the cell so transfected in culture mediumunder conditions such that full length soluble protein C or biologicallyactive polypeptide variants of protein C, is secreted. Further, the cellis a eukaryotic cell, e.g. mammalian cell such as Syrian hamster AV12cell, human embryonic 293 cell, or Baby Hamster Kidney cell.

The protein C used in the treatment of viral hemorrhagic fever can beformulated according to known methods to prepare pharmaceutically usefulcompositions. For example, a desired formulation would be one that is astable lyophilized product of high purity comprising a bulking agentsuch as sucrose, a salt such as sodium chloride, a buffer such as sodiumcitrate and protein C or aPC.

The protein C will be administered parenterally to ensure its deliveryinto the bloodstream in an effective form by injecting the appropriatedose as continuous infusion for about 1 hour to about 240 hours.

Those skilled in the art can readily optimize pharmaceutically effectivedosages and administration regimens for therapeutic compositionscomprising protein C, as determined by good medical practice and theclinical condition of the individual patient. Generally, the amount ofprotein C administered will be from about 5.0 μg/kg/hr to about 250μg/kg/hr. Preferably, the protein C used in the treatment of viralhemorrhagic fever is activated protein C (aPC). The amount of aPCadministered will be from about 1.0 μg/kg/hr to about 96 μg/kg/hr. Morepreferably the amount of aPC administered will be about 1.0 μg/kg/hr toabout 50 μg/kg/hr. While more preferably the amount of aPC administeredwill be about 1.0 μg/kg/hr to about 35 μg/kg/hr. Even more preferablythe amount of aPC administered will be about 5.0 μg/kg/hr to about 30μg/kg/hr. Yet even more preferably the amount of aPC administered willbe about 15 μg/kg/hr to 30 μg/kg/hr. Still even more preferably theamount of aPC administered will be about 20 μg/kg/hr to 30 μg/kg/hr. Thepreferable amount of aPC administered will be about 24 μg/kg/hr. Themost preferable amount of aPC administered will be about 48 μg/kg/hr.The appropriate dose of aPC administered will result in a reduction ofthe thrombotic complications associated with viral hemorrhagic fever.

The plasma ranges obtained from the amount of aPC administered will beabout 2 ng/ml to about 300 ng/ml. The preferred plasma ranges are fromabout 2 ng/ml to 200 ng/ml. Most preferably, plasma ranges are fromabout 30 ng/ml to about 150 μg/ml and still more preferably about 100ng/ml.

Alternatively, the aPC will be administered by injecting one third ofthe appropriate dose per hour as a bolus injection followed by theremaining two thirds of the hourly dose as continuous infusion for onehour followed by continuous infusion of the appropriate dose fortwenty-three hours which results in the appropriate dose administeredover 24 hours. In addition, the bolus injection will be administered viaan intravenous bag drip pump or syringe pump at 2 times the normal ratefor 15 minutes followed by 1.5 times the normal rate for 45 minutes. Thenormal rate i.e. that rate which has been determined to administer theappropriate dose level of the therapeutic agent per time period, is thencontinued for up to 240 hours.

The use of protein C in the treatment of viral hemorrhagic fever aspresented in the present invention will provides a needed therapy for aserious and debilitating disorder. The use of protein C is efficaciousand avoids complications such as bleeding tendency, toxicity, and othergeneral side effects of currently available anti-coagulant agents.

The following examples are provided merely to further illustrate thepresent invention. The scope of the invention shall not be construed asmerely consisting of the following examples.

PREPARATION 1 Preparation of Human Protein C

Recombinant human protein C (r-hPC) was produced in Human Kidney 293cells by techniques well known to the skilled artisan such as those setforth in Yan, U.S. Pat. No. 4,981,952, the entire teaching of which isherein incorporated by reference. The gene encoding human protein C isdisclosed and claimed in Bang, et al., U.S. Pat. No. 4,775,624, theentire teaching of which is incorporated herein by reference. Theplasmid used to express human protein C in 293 cells was plasmid PLPCwhich is disclosed in Bang, et al., U.S. Pat. No. 4,992,373, the entireteaching of which is incorporated herein by reference. The constructionof plasmid pLPC is also described in European Patent Publication No. 0445 939, and in Grinnell, et al., 1987, Bio/Technology 5:1189-1192, theteachings of which are also incorporated herein by reference. Briefly,the plasmid was transfected into 293 cells, then stable transformantswere identified, subcultured and grown in serum-free media.

After fermentation, cell-free medium was obtained by microfiltration.

The human protein C was separated from the culture fluid by anadaptation of the techniques of Yan, U.S. Pat. No. 4,981,952. Theclarified medium was made 4 mM in EDTA before it was absorbed to ananion exchange resin (Fast-Flow Q, Pharmacia). After washing with 4column volumes of 20 mM Tris, 200 mM NaCl, pH 7.4 and 2 column volumesof 20 mM Tris, 150 mM NaCl, pH 7.4, the bound recombinant human proteinC zymogen was eluted with 20 mM Tris, 150 mM NaCl, 10 mM CaCl₂, pH 7.4.The eluted protein was greater than 95% pure after elution as judged bySDS-polyacrylamide gel electrophoresis.

Further purification of the protein was accomplished by making theprotein 3 M in NaCl followed by adsorption to a hydrophobic interactionresin (Toyopearl Phenyl 650 M, TosoHaas) equilibrated in 20 mM Tris, 3 MNaCl, 10 mM CaCl₂, pH 7.4. After washing with 2 column volumes ofequilibration buffer without CaCl₂, the recombinant human protein C waseluted with 20 mM Tris, pH 7.4.

The eluted protein was prepared for activation by removal of residualcalcium. The recombinant human protein C was passed over a metalaffinity column (Chelex-100, Bio-Rad) to remove calcium and again boundto an anion exchanger (Fast Flow Q, Pharmacia). Both of these columnswere arranged in series and equilibrated in 20 mM Tris, 150 mM NaCl, 5mM EDTA, pH 7.4. Following loading of the protein, the Chelex-100 columnwas washed with one column volume of the same buffer beforedisconnecting it from the series. The anion exchange column was washedwith 3 column volumes of equilibration buffer before eluting the proteinwith 0.4 M NaCl, 20 mM Tris-acetate, pH 6.5. Protein concentrations ofrecombinant human protein C and recombinant activated protein Csolutions were measured by UV 280 nm extinction E^(0.1%=)1.81 or 1.85,respectively.

PREPARATION 2 Activation of Recombinant Human Protein C

Bovine thrombin was coupled to Activated CH-Sepharose 4B (Pharmacia) inthe presence of 50 mM HEPES, pH 7.5 at 4° C. The coupling reaction wasdone on resin already packed into a column using approximately 5000units thrombin/mL resin. The thrombin solution was circulated throughthe column for approximately 3 hours before adding 2-amino-ethanol (MEA)to a concentration of 0.6 mL/L of circulating solution. TheMEA-containing solution was circulated for an additional 10-12 hours toassure complete blockage of the unreacted amines on the resin. Followingblocking, the thrombin-coupled resin was washed with 10 column volumesof 1 M NaCl, 20 mM Tris, pH 6.5 to remove all non-specifically boundprotein, and was used in activation reactions after equilibrating inactivation buffer.

Purified r-hPC was made 5 mM in EDTA (to chelate any residual calcium)and diluted to a concentration of 2 mg/mL with 20 mM Tris, pH 7.4 or 20mM Tris-acetate, pH 6.5. This material was passed through a thrombincolumn equilibrated at 37° C. with 50 mM NaCl and either 20 mM Tris pH7.4 or 20 mM Tris-acetate pH 6.5. The flow rate was adjusted to allowfor approximately 20 min. of contact time between the r-hPC and thrombinresin. The effluent was collected and immediately assayed for amidolyticactivity. If the material did not have a specific activity (amidolytic)comparable to an established standard of protein C, it was recycled overthe thrombin column to activate the r-hPC to completion. This wasfollowed by 1:1 dilution of the material with 20 mM buffer as above,with a pH of either 7.4 or 6.5 to keep the protein C at lowerconcentrations while it awaited the next processing step.

Removal of leached thrombin from the protein C material was accomplishedby binding the protein C to an anion exchange resin (Fast Flow Q,Pharmacia) equilibrated in activation buffer (either 20 mM Tris, pH 7.4or 20 mM Tris-acetate, pH 6.5) with 150 mM NaCl. Thrombin does notinteract with the anion exchange resin under these conditions, butpasses through the column into the sample application effluent. Once theprotein C is loaded onto the column, a 2-6 column volume wash with 20 mMequilibration buffer is done before eluting the bound protein C with astep elution using 0.4 M NaCl in either 5 mM Tris-acetate, pH 6.5 or 20mM Tris, pH 7.4. Higher volume washes of the column facilitated morecomplete removal of the dodecapeptide. The material eluted from thiscolumn was stored either in a frozen solution (−20° C.) or as alyophilized powder.

The anticoagulant activity of activated protein C was determined bymeasuring the prolongation of the clotting time in the activated partialthromboplastin time (APTT) clotting assay. A standard curve was preparedin dilution buffer (1 mg/mL radioimmunoassay grade bovine serum albumin[BSA], 20 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN₃) ranging in protein Cconcentration from 125-1000 ng/mL, while samples were prepared atseveral dilutions in this concentration range. To each sample cuvette,50 μL of cold horse plasma and 50 μL of reconstituted activated partialthromboplastin time reagent (APTT Reagent, Sigma) were added andincubated at 37° C. for 5 min. After incubation, 50 μL of theappropriate samples or standards were added to each cuvette. Dilutionbuffer was used in place of sample or standard to determine basalclotting time. The timer of the fibrometer (CoA Screener HemostasisAnalyzer, American Labor) was started immediately after the addition of50 μL 37° C. 30 mM CaCl₂ to each sample or standard. Activated protein Cconcentration in samples are calculated from the linear regressionequation of the standard curve. Clotting times reported here are theaverage of a minimum of three replicates, including standard curvesamples.

The above descriptions enable one with appropriate skill in the art toprepare protein C for utilization in the treatment of viral hemorrhagicfever.

PREPARATION 3 Formulation of Activated Protein C

A stable lyophilized formulation of activated protein C was prepared bya process which comprises lyophilizing a solution comprising about 2.5mg/mL activated protein C, about 15 mg/mL sucrose, about 20 mg/mL NaCl,and a sodium citrate buffer having a pH greater than 5.5 but less than6.5. Additionally, the stable lyophilized formulation of activatedprotein C comprises lyophilizing a solution comprising about 5 mg/mLactivated protein C, about 30 mg/mL sucrose, about 38 mg/mL NaCl, and acitrate buffer having a pH greater than 5.5 but less than 6.5.

The ratio of protein C:salt:bulking agent (w:w:w) is an important factorin a formulation suitable for the freeze drying process. The ratiovaries depending on the concentration of protein C, salt selection andconcentration and bulking agent selection and concentration.Particularly, a ratio of about 1 part activated protein C to about 7.6parts salt to about 6 parts bulking agent is preferred.

A unit dosage formulation of activated protein C suitable foradministration by continuous infusion was prepared by mixing activatedprotein C, NaCl, sucrose, and sodium citrate buffer. After mixing, 4 mLof the solution was transferred to a unit dosage receptacle andlyophilized.

The unit dosage receptacle containing about 5 mg to about 20 mg ofactivated protein C, suitable for administering a dosage of about 0.01mg/kg/hr to about 0.05 mg/kg/hr to patients in need thereof, was sealedand stored until use.

EXAMPLE 1 A Placebo-controlled Double Blind Trial of Recombinant HumanActivated Protein C (r-aPC) in Patients with Viral Hemorrhagic Fever

Studies of viral hemorrhagic fever patients have demonstratedabnormalities of platelet survival and aggregation as well asalterations in clotting factors. The current treatment approach topatients with viral hemorrhagic fever is primarily supportive in theabsence of an effective anti-viral agent.

This trial aims to show that the infusion of r-aPC results in astatistically significant reduction in mortality associated with viralhemorrhagic fever.

Inclusion criteria include patients with viral hemorrhagic fever. Thesepatients are entered into the trial immediately upon diagnosis and entryinto the hospital. Patients meeting the inclusion criteria for viralhemorrhagic fever are given the standard supportive care. In addition,the patients receive either placebo or r-aPC for 96 hours. r-aPC isgiven in a dose of 24 μg/kg/hr.

The primary endpoints of the study are: the safety and efficacy of r-aPCas compared to placebo, and; the ability of r-aPC to correctcoagulopathy and effect mortality.

We claim:
 1. A method of treating a patient suffering from viralhemorrhagic fever which comprises, administering to said patient apharmaceutically effective amount of activated protein C.
 2. The methodaccording to claim 1, wherein the amount of human activated protein C isabout 1 μg/kg/hr to about 96 μg/kg/hr.
 3. The method of claim 2, whereinthe human activated protein C is administered by continuous infusion forabout 1 to about 240 hours.
 4. A method of treating viral hemorrhagicfever in a patient in need thereof, which comprises administering tosaid patient a pharmaceutically effective amount of activated protein Csuch that an activated protein C plasma level of about 2 ng/ml to about300 ng/ml is achieved.
 5. The method of claim 4 wherein the activatedprotein C is administered in a bolus injection.
 6. The method of claim 4wherein the activated protein C is administered by continuous infusionfor about 1 to about 240 hours.
 7. The method of claim 4 wherein theactivated protein C is administered first as a bolus then as acontinuous infusion.
 8. The method of claim 7 wherein one third of theactivated protein C required to achieve activated protein C plasmalevels in the range of about 2 ng/ml to about 300 ng/ml is administeredin a bolus injection followed by continuous infusion of the remainingtwo thirds of the activated protein C.